Recent large-scale genetic screens have uncovered a multitude of genes implicated in brain development, learning and memory, regeneration, and neurological diseases. Determining the function of these genes in vivo necessitates advanced techniques for controlling the timing and location of gene expression, combined with specific assays of brain cell function or morphology. In some cases it will be important to introduce genes into postmitotic cells which are refractory to most available gene transfer methods. In other cases, introduction of genes of interest into pluripotent cells such as stem cells will be required to determine their function or to provide therapeutic value.
Morphologic studies of neuronal development in Xenopus following viral gene transfer and in transgenic Drosophila exemplify the potential for elucidating gene function when the genetic makeup of individual neurons can be manipulated and the consequences of the gene transfer on neuronal structure can be observed by in vivo imaging techniques (Li, et al., Nature Neurosci 3, 217-225 (2000), Nedivi, et al., Science 281, 1863-1866 (1998), Wu G-Y and Cline H T, Science 279, 222-226 (1998); Zou D-J and Cline H T, Neuron 16; 529-539 (1996), Davis, et al., Neuron 19, 561-73 (1997)). As another example, side-by-side comparison of the synaptic properties of individual genetically modified neurons with unaffected neurons in the same brain provides a powerful tool for the elucidation of gene function (Hayashi, et al., Science 287, 2262-2267 (2000). However, current techniques for spatially and temporally controlled introduction of genes of interest into single cells within central nervous system tissue is limited. One versatile technique applicable to this problem is electroporation.
Electroporation is a popular technique for introducing macromolecules, including DNA, RNA, dyes, proteins and various chemical agents, into cells. Electroporation refers to the permeabilization of cell membranes by application of short duration electric field pulses, traditionally between relatively large plate electrodes (Neumann, et al., Bioelectrochem Bioenerg 48, 3-16 (1999); Ho, et al., Crit Rev Biotechnol 16, 349-62 (1996)). Large external electric fields induce high transmembrane potentials leading to the formation of minute pores (20-120 nm diameter) restricted to small regions of the cell membrane (<0.1%) adjacent to the electrodes. During the electric pulse, charged macromolecules, including DNA, are actively transported by electrophoresis across the cell membrane through these pores (Neumann, et al., Biophys J 71, 868-77 (1996)). Noncharged molecules can also enter through the pores by passive diffusion. Upon pulse termination, pores reseal over hundreds of milliseconds as measured by recovery of normal membrane conductance values (Ho, 1996, supra).
Although electroporation is an established method for implantation of exogenous materials in various cell types including both prokaryotic and eukaryotic cells, e.g., transformation and transfection of cell types ranging from bacteria to mammalian cell lines, its application to brain cells such as neurons and glial cells in intact tissues has been relatively limited. Electroporation has been successful for gene transfer into large numbers of neurons in chick (Atkins, et al., Biotechniques 28, 94-6, 98, 100 (2000); Sakamoto, et al., FEBS Lett 426, 337-41 (1998); Koshiba-Takeuchi, et al. Science 287, 134-1 (2000)) and mouse embryos (Akamatsu, et al., Proc Natl Acad Sci USA 96, 9885-90 (1999); Miyasaka, et al., Neuroreport 10, 2319-23 (1999)). Electroporation is being utilized for neuronal gene transfer since it has many advantages over more common transfection methods including viral gene transfer, gene gun biolistics, lipofection and microinjection. Electroporation lacks the potentially toxic effects of viruses and lipofection and the potential physical damage due to the biolistic gene gun and microinjection. Electroporation is also significantly more efficient than either lipofection, microinjection or gene gun biolistics, in terms of numbers of transfected cells and the intensity of foreign gene expression (See Muramatsu, et al., Biochem Biophys Res Commun 230, 376-80 (1997)). Few viruses are available which can infect postmitotic brain cells. Viruses are also limited by the size of the foreign DNA that can be inserted into their genome and thereby transferred to cells. This limits the use of viral gene transfer to study the function of large genes and the use of dicistronic complexes to introduce multiple genes into cells. Finally, lipofection can only be used in proliferating cells and results in transient expression lasting only a few days.
In situations where single-cell gene transfer in intact tissues is desired to express multiple genes, current transfection techniques are insufficient. For many applications, co-expression of multiple genes is desired to allow one to visually identify the transfected cells or to study the interaction of multiple proteins. Co-expression of multiple genes may also be required for assays of brain cell structure following expression of a fluorescent protein along with a gene of interest. Low titre virus can be used to sparsely infect cells, but viral infection is subject to the limitations mentioned above. Although gene-gun biolistics may be utilized to deliver multiple genes, it cannot be used in vivo and is thus limited in its application. Gene transfer by microinjection typically requires that DNA be delivered into the cell nucleus. This method is only applicable to cells plated on a coverslip in which a micropipette can be visualized as it penetrates the nucleus using expensive microscopy equipment. While electroporation is a promising transfection technique, precise targeting is not feasible using traditional, large electrodes. However, one of the powerful attributes of electroporation is the ability to localize transfection by controlling exposure to either DNA or the electric field.